GSM7179134: GEX patient3_scACTP_1; Homo sapiens; RNA-Seq
TILs were generated in the GMP manufacturing facilities of the Centre of Thérapies Expérimentales (CTE) of the Centre Hospitalier Universitaire Vaudois (CHUV) under specific conditions previously described (Gannon et al., Cytotherapy, 2020). Some TILs of the infusion product were cryopreserved in 90% human serum + 10% DMSO for translational studies. The day of the assay, the samples of the infused T cell product were thawed and TILs undergone overnight recovery in R8 medium supplemented with 3000 IU/mL IL-2 (Proleukin) or fresh TILs were directly used. TILs were resuspended in PBS + 0.04% BSA and DAPI (Invitrogen) staining was performed. Live cells were sorted with a BD FACS Melody sorter and manually counted to assess viability with Trypan blue. Live TILs were resuspended at a density of 600-1200 cells µL-1 with a viability of >90% and subjected to a 10X Chromium instrument for single-cell analysis. The standard protocol of 10X Genomics was followed and the reagents for the Chromium Single Cell 5' Library and V(D)J library (v1.0 or v1.1 Chemistry) were used. 15'000 cells were loaded per sample. Using a microfluidic technology, single-cell were captured and lysed, mRNA was reverse transcribed to barcoded cDNA using the provided reagents (10X Genomics). 14 PCR cycles were used to amplify cDNA and the final material was divided into two fractions: first fraction was target-enriched for TCRs and V(D)J library was obtained according to manufacturer protocol (10X Genomics). The second fraction was processed for 5' gene expression library following the manufacturer's instruction (10X Genomics). Barcoded V(D)J libraries and GEX libraries were pooled and sequenced by an Illumina HiSeq 4000 sequencer.